The invention relates to a technique for a diagnosis of a specific disease, and in particular, to a process for screening for a gene involving a coronary artery spasm-associated disease.
Intrinsic nitric oxide (intrinsic NO) is a substance which recently has been identified in vivo, and has been shown to act as a vasodilator (18-25), a neurotransmitter (11, 12) or an immunoreactant (13-17). The intrinsic NO is synthesized from L-arginine due to a family consisting of at least three nitric oxide synthases (NOSs), i.e., neural NOS, endothelial NOS, and macrophage NOS. Neural NOS and endothelial NOS are constitutive, and enhanced in their activity depending on concentrations of intracellular calcium. On the other hand, macrophage NOS is independent of calcium, and activated by inflammation. Early, some researchers determined the gene structure of endothelial cell nitric oxide synthase (eNOS), and reported that the eNOS gene locates at 7q35-36 in chromosome, and is composed of 26 exons, and spans 21 kb (18-25).
Coronary artery spasm is understood to be a pathogenic factor of various ischemic heart diseases such as coronary spastic angina and variant angina (1-6). However, a mechanism of the coronary spasm is not known well.
Thus, the present inventors have carried out investigations for clarifying the mechanism of the coronary spasm, considering that such investigations may contribute to development of an early diagnosis and treatment of the ischemic diseases to be associated with the coronary spasm. It should be noted that most of the previous studies have pointed out that a hyper-contractility of smooth muscle is more important in the pathogenic mechanism of the coronary spasm rather than a damage of hemangioendothelium (35).
Acetylcholine generally acts on smooth muscle to contract it, but acetylcholine eventually may lead vasodilatation by means of secretion of nitric oxide (NO) acting on smooth muscle, when the endothelium is uninjured. However, coronary administration of acetylcholine into patients having coronary spastic angina induces the coronary spasm rather than vasodilatation (7,8). In connection with this fact, the present inventors previously have demonstrated that the basal secretion of NO caused by acetylcholine is low in the patients having coronary spastic angina (10).
Since nitroglycerin and a nitrous acid medicine can effect vasodilatation in the course of forming NO in vivo the basal secretion of NO might be decreased in the coronary artery of the patients having coronary spastic angina, which is supported by the fact that the reaction of vessel in which the NO synthesis is suppressed, to the nitrous acid medicine is supersensitive (9), and the coronary artery in the patients having coronary spastic angina is supersensitive to nitroglycerin. These results would suggest the possibility of a strong relationship between coronary spasm and NO, specifically the endothelium where NO is secreted. To date, no one has paid attention to the relationship between coronary spasm and the endothelium where NO is secreted, except under the circumstance that the decrease in basal secretion of NO is known.
From a different point of view, the present inventors have noticed that coronary spasm may be caused by a genetic background in the light of the fact that coronary spasm is more frequent in Japan than in United States of America and Europe (26, 27).
On the basis of the above findings, the present inventors have focused on a gene of one of NOS enzymes which produce intrinsic NO, i.e., endothelial cell nitric oxide synthase (eNOS), examined if the gene is responsible for a development of coronary spasm, and finally completed the present invention.
The present invention relates to a process for screening for a gene involving a coronary artery spasm-associated disease, which comprises (obtaining DNA sample from a subject tested for a coronary artery spasm-associated disease, and) detecting the presence of one or more nucleotide changes in the gene of endothelial nitric oxide synthase (eNOS), which changes are selected from a group consisting of the changes from guanine (G) to thymine (T) at position 894 and from cytosine (C) to thymine (T) at position 774 of the cDNA sequence of the eNOS gene (of SEQ ID NO:2), and the changes from thymine (T) to cytosine (C) at position xe2x88x92786, from adenine (A) to guanine (G) at position xe2x88x92922 and from thymine (T) to adenine (A) at position xe2x88x921468 of the 5xe2x80x2-flanking region of the eNOS gene, as well as the changes at the corresponding positions in the complementary strand thereof. Preferably, the present invention relates to the above process which comprises detecting the presence of two or more nucleotide changes selected from any one of nucleotide changes in the eNOS gene, and any one of nucleotide changes in the 5xe2x80x2-flanking region thereof, and more preferably, to the above process in which the coronary artery spasm-associated disease is angina, and further preferably, to the above process in which the coronary artery spasm-associated disease is coronary spastic angina.
In one aspect, the present invention relates to the process for screening for the above changes which comprises RFLP method wherein the presence or absence of a cleavage caused by a restriction enzyme is detected. Particularly, the present invention relates to the process in which the detection of the presence of the changes is performed by amplifying the relevant region to exon 7 in the cDNA coding eNOS by means of PCR, digesting the amplified fragments with the restriction enzyme(s) BanII and/or MboI, and electrophoresing the fragments digested with the enzyme, as well as the process in which the detection of the presence of the changes in exon 6 is performed by treating with the restriction enzyme FokI, the process in which the detection of the presence of the changes at position xe2x88x92786 in the 5xe2x80x2-flanking region of the eNOS gene by treating with the restriction enzyme MspI, and the process in which the detection of the presence of the changes at position xe2x88x921468 in the 5xe2x80x2-flanking region of the eNOS gene by treating with the restriction enzyme MboI.
In one aspect, the present invention relates to a kit for performing the process of the present invention, and specifically, to a kit for diagnosing a coronary artery spasm-associated disease, which comprises (a pair of) amplification (oligonucleotides) for amplifying the relevant portion to exon 7 in the cDNA coding eNOS by PCR, and the restriction enzyme(s) BanII and/or MboI; a kit for diagnosing a coronary artery spasm-associated disease, which comprises an amplification oligonucleotide for amplifying the relevant portion to exon 6 in the cDNA coding eNOS by PCR, and the restriction enzyme FokI; a kit for diagnosing a coronary artery spasm-associated disease, which comprises an amplification oligonucleotide for amplifying a portion encompassing the xe2x88x92786 position in the 5xe2x80x2-flanking region of the eNOS gene by PCR, and the restriction enzyme MspI; and a kit for diagnosing a coronary artery spasm-associated disease, which comprises an amplification oligonucleotide for amplifying a portion encompassing the xe2x88x921468 position in the 5xe2x80x2-flanking region of the eNOS gene by PCR, and the restriction enzyme MboI. It should be noted that the amplification oligonucleotide contained in the kit of the present invention includes oligonucleotides other than those listed in hereinafter Table 1, and can be selected among any oligonucleotides by reference to the genome sequence of eNOS.
In one aspect, the present invention relates to a method for diagnosing a coronary artery spasm-associated disease, which comprises detecting the presence of any one or more nucleotide changes in the eNOS gene as defined above which are responsible for the coronary artery spasm-associated disease.